Phytoglycogen and glycogen are polysaccharides of glucose composed of α-1,4-glucan chains, highly branched via α-1,6-glucosidic linkages, which function as energy storage mediums in plant and animal cells. Glycogen is present in animal tissue in the form of dense particles with diameters of 20-200 nm. Glycogen is also found to accumulate in microorganisms, e.g., bacteria and yeasts. Phytoglycogen is a polysaccharide that is similar to glycogen, both in terms of its structure and physical properties and originates in plants.
Glycogen and phytoglycogen are considered “highly polydisperse” or heterogeneous materials. Glycogen typically has a molecular weight between 106 and 108 Daltons with a corresponding large polydispersity for known preparations. Transmission electron microscopy (TEM) observations of animal and plant tissues and extracted glycogen/phytoglycogen preparations have revealed the particulate nature of these polysaccharides. Commonly reported glycogen or phytoglycogen particle diameters are in the range of 20-300 nm and have either continuous or multimodal size distribution. Small, 20-30 nm, particles are termed β-particles and large, 100-300 nm—α-particles. The α-particles are considered to be composed of β-particles as a result of aggregation or clustering [1].
Various methods have been developed to isolate glycogen and phytoglycogen from living organisms, most often for the purpose of quantifying the amount of total glycogen accumulated in biological samples, and, infrequently, for the purpose of using the glycogen as a product in applications.
The most frequently used method is extraction from animal tissues, particularly from marine animals, especially mollusks, because of their ability to accumulate glycogen. For example, U.S. Pat. No. 5,734,045 discloses a process for the preparation of protein-free glycogen from mussels by using hot alkali extraction, following neutralization and treatment of the resulting solution with cationic resins. Glycogen can also be produced via fermentation of yeasts as described, for example, in patent application WO/1997/021828. U.S. Pat. No. 7,670,812 describes a process for the biosynthetic production of glycogen-like polysaccharides by exposing a mixture of enzymes to low molecular weight dextrins. Sweet corn and sweet rice can be used as a source of glycogen; see, for example, patent application EP0860448B1, which describes a process of isolating glycogen from the kernels of sweet rice.
The main steps of glycogen/phytoglycogen isolation typically include: biomass disintegration via pulverization/grinding/milling etc.; glycogen extraction into water phase; separation of insoluble solid particles via filtration and/or centrifugation; elimination of finely dispersed or solubilized lipids, proteins and low molecular weight contaminates; and concentration and drying.
To increase the yield of glycogen in the second extraction step, extraction is often performed at elevated temperatures and/or using alkaline or acidic solutions. Such procedures include initial treatment of ground biological material with hot concentrated (20-40%) solution of alkali [2, 3], cold acids [4] or boiling water [5].
The procedures used in the conventional methods of glycogen isolation/purification result in considerable hydrolysis of the glycogen structure, with significant increases of lower molecular weight products and chemical alteration of the molecule.
Various milder extraction procedures have been developed, such as cold water extraction [6], and resulting products were claimed to be close representation of natural state of the glycogen. However, known glycogen preparations produced by cold water extraction method are highly polydisperse [7,1].
Various methods are known for performing the step of purifying crude glycogen extract from finely dispersed proteins, lipids, nucleic acids, and other polysaccharides. Protein and nucleic acids can be removed via selective precipitation with deoxycholate (DOC) trichloroacetic acid (TCA), polyvalent cations, and/or enzymatic (protease, nuclease) treatment. Also methods of removing proteins by salting them out (e.g., with ammonium sulfate), or by ion-exchange have been used. Another common method of protein removal is thermal coagulation, normally at 65-100° C., following by centrifugation or filtration. Autoclaving (121° C. at 1 atm) has also been used to coagulate proteins in phytoglycogen extract [8]. Furthermore, proteins and lipids can be removed with phenol-water extraction.
International patent application publication no. WO 2013/019977 (Yao) teaches a method for obtaining extracts that include phytoglycogen that includes ultrafiltration, but also subjecting the aqueous extract to enzymatic treatment that degrades both phytoglycogen as well as other polysaccharides. Yao provides a method to reduce viscosity of phytoglycogen material by subjecting it to beta-amylolysis, i.e., enzymatic hydrolysis using beta-amylase. The “purified phytoglycogen” materials yielded by the methods of Yao include not only phytoglycogen, but derivatives of phytoglycogen, including beta-dextrins and the digestion products of other polysaccharides. The method of Yao further involves heating the extract to 100° C. (see Yao Example 1).
U.S. Pat. No. 5,895,686 discloses a method for extracting phytoglycogen from rice by water or a water-containing solvent (at room temperature) and the removal of proteins by thermal denaturation and TCA precipitation. The product has a multimodal molecular weight distribution, with correspondingly high polydispersity, and large water solution viscosities. These properties can be attributed to the presence of substantial amounts of amylopectin and amylose in glycogen preparations from plant material, but also to glycogen degradation during the glycogen extraction process.
U.S. Pat. Nos. 5,597,913 and 5,734,045 describe procedures that result in glycogen that is substantially free of nitrogenous compounds and reducing sugars as an indication of its purity from proteins and nucleic acids. These patents teach the use of boiling of the selected tissues in solutions of high pH.
United States patent application publication no. United States 20100272639 A1, assigned to the owner of the present invention, provides a process for the isolation of glycogen from bacterial and shell fish biomass. Bacteria is taught as preferred since the process can be conducted to yield a biomass that does not have other large molecular weight polysaccharides such as amylopectin and amylose and is free of pathogenic bacteria, parasites, viruses and prions associated with shell-fish or animal tissue. The processes disclosed generally include the steps of cell disintegration by French pressing, or by chemical treatment; separation of insoluble cell components by centrifugation; elimination of proteins and nucleic acids from cell lyzate by enzymatic treatment followed by dialysis which produces an extract containing crude polysaccharides and lypopolysaccharides (LPS) or, alternatively, phenol-water extraction; elimination of LPS by weak acid hydrolysis, or by treatment with salts of multivalent cations, which results in the precipitation of insoluble LPS products; and purification of the glycogen enriched fraction by ultrafiltration and/or size exclusion chromatography; and precipitation of glycogen with a suitable organic solvent or a concentrated glycogen solution can be obtained by ultrafiltration or by ultracentrifugation; and freeze drying to produce a powder of glycogen. Glycogen isolated from bacterial biomass was characterized by MWt 5.3-12.7×106 Da, had particle size 35-40 nm in diameter and were monodisperse (PDI=Mn/Mw=1.007-1.03).